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Reproduction (Cambridge, England) Aug 2024Ovarian aging results in reactive oxygen species accumulation and mitochondrial deterioration. During the aging process, GRSF1 deficiency attenuates mitochondrial...
IN BRIEF
Ovarian aging results in reactive oxygen species accumulation and mitochondrial deterioration. During the aging process, GRSF1 deficiency attenuates mitochondrial function in aging granulosa cells.
ABSTRACT
Ovarian aging critically influences reproductive potential, with a marked decrease in oocyte quality and quantity and an increase in oxidative stress and mitochondrial dysfunction. This study elucidates the role of guanine-rich RNA sequence binding factor 1 (GRSF1) in the aging of ovarian granulosa cells (GCs). We observed a significant reduction in GRSF1 within GCs correlating with patient age, utilizing clinical samples from IVF patients. Using an siRNA-mediated knockdown technique, we established that diminished GRSF1 expression exacerbates mitochondrial dysfunction, elevates reactive oxygen species, and impairs ATP production. Furthermore, RNA immunoprecipitation revealed GRSF1's interaction with superoxide dismutase 2 (SOD2) mRNA, a key antioxidant enzyme, suggesting a mechanism whereby GRSF1 modulates oxidative stress. Downregulation of SOD2 reversed the protective effects of GRSF1 overexpression on mitochondrial function. These insights into the role of GRSF1 in ovarian aging may guide the development of interventions to improve fertility outcomes in advanced age.
Topics: Female; Granulosa Cells; Humans; Mitochondria; Reactive Oxygen Species; Oxidative Stress; Aging; Cellular Senescence; Adult; Superoxide Dismutase; Cells, Cultured; Poly(A)-Binding Proteins
PubMed: 38819377
DOI: 10.1530/REP-24-0015 -
Chembiochem : a European Journal of... May 2024RNA labeling is an invaluable tool for investigation of the function and localization of nucleic acids. Labels are commonly incorporated into 3' end of RNA and the...
RNA labeling is an invaluable tool for investigation of the function and localization of nucleic acids. Labels are commonly incorporated into 3' end of RNA and the primary enzyme used for this purpose is RNA poly(A) polymerase (PAP), which belongs to the class of terminal nucleotidyltransferases (NTases). However, PAP preferentially adds ATP analogs, thus limiting the number of available substrates. Here, we report the use of another NTase, CutA from the fungus Thielavia terrestris. Using this enzyme, we were able to incorporate into the 3' end of RNA not only purine analogs, but also pyrimidine analogs. We engaged strain-promoted azide-alkyl cycloaddition (SPAAC) to obtain fluorescently labeled or biotinylated transcripts from RNAs extended with azide analogs by CutA. Importantly, modified transcripts retained their biological properties. Furthermore, fluorescently labeled mRNAs were suitable for visualization in cultured mammalian cells. Finally, we demonstrate that either affinity studies or molecular dynamic (MD) simulations allow for rapid screening of NTase substrates, what opens up new avenues in the search for the optimal substrates for this class of enzymes.
PubMed: 38818670
DOI: 10.1002/cbic.202400202 -
Science China. Life Sciences Jun 2024Generally shortened 3' UTR due to alternative polyadenylation (APA) is widely observed in cancer, but its regulation mechanisms for cancer are not well characterized....
Generally shortened 3' UTR due to alternative polyadenylation (APA) is widely observed in cancer, but its regulation mechanisms for cancer are not well characterized. Here, with profiling of APA in colorectal cancer tissues and poly(A) signal editing, we firstly identified that the shortened 3' UTR of CTNNIBP1 in colorectal cancer promotes cell proliferation and migration. We found that liquid-liquid phase separation (LLPS) of PABPN1 is reduced albeit with higher expression in cancer, and the reduction of LLPS leads to the shortened 3' UTR of CTNNBIP1 and promotes cell proliferation and migration. Notably, the splicing factor SNRPD2 upregulated in colorectal cancer, can interact with glutamic-proline (EP) domain of PABPN1, and then disrupt LLPS of PABPN1, which attenuates the repression effect of PABPN1 on the proximal poly(A) sites. Our results firstly reveal a new regulation mechanism of APA by disruption of LLPS of PABPN1, suggesting that regulation of APA by interfering LLPS of 3' end processing factor may have the potential as a new way for the treatment of cancer.
Topics: Humans; Colorectal Neoplasms; Cell Proliferation; Poly(A)-Binding Protein I; Cell Movement; Polyadenylation; 3' Untranslated Regions; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Phase Separation
PubMed: 38811444
DOI: 10.1007/s11427-023-2495-x -
BMC Genomics May 2024Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing...
BACKGROUND
Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been expanding the potential of dRNA-seq, its sequencing accuracy and error patterns remain understudied.
RESULTS
We present the first comprehensive evaluation of sequencing accuracy and characterisation of systematic errors in dRNA-seq data from diverse organisms and synthetic in vitro transcribed RNAs. We found that for sequencing kits SQK-RNA001 and SQK-RNA002, the median read accuracy ranged from 87% to 92% across species, and deletions significantly outnumbered mismatches and insertions. Due to their high abundance in the transcriptome, heteropolymers and short homopolymers were the major contributors to the overall sequencing errors. We also observed systematic biases across all species at the levels of single nucleotides and motifs. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines and adenines. By examining raw signal data, we identified the underlying signal-level features potentially associated with the error patterns and their dependency on sequence contexts. While read quality scores can be used to approximate error rates at base and read levels, failure to detect DNA adapters may be a source of errors and data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (basecalling) artefacts. Lastly, we generated dRNA-seq data using the latest SQK-RNA004 sequencing kit released at the end of 2023 and found that although the overall read accuracy increased, the systematic errors remain largely identical compared to the previous kits.
CONCLUSIONS
As the first systematic investigation of dRNA-seq errors, this study offers a comprehensive overview of reproducible error patterns across diverse datasets, identifies potential signal-level insufficiency, and lays the foundation for error correction methods.
Topics: Sequence Analysis, RNA; Nanopore Sequencing; Nanopores; Humans; Animals; RNA; High-Throughput Nucleotide Sequencing
PubMed: 38807060
DOI: 10.1186/s12864-024-10440-w -
Chemical Communications (Cambridge,... Jun 2024A programmably engineered stochastic RNA nanowalker powered by duplex-specific nuclease (DSN) is developed. By utilizing poly-adenine-based spherical nucleic acids...
A programmably engineered stochastic RNA nanowalker powered by duplex-specific nuclease (DSN) is developed. By utilizing poly-adenine-based spherical nucleic acids (polyA-SNA) to accurately regulate the densities of DNA tracks, the nanowalker showcases its capability to identify miRNA-21, miRNA-486, and miRNA-155 with quick kinetics and attomolar sensitivity, positioning it as a promising option for cancer clinical surveillance.
Topics: MicroRNAs; Humans; Nanostructures; Poly A; DNA; Stochastic Processes; Biosensing Techniques
PubMed: 38804211
DOI: 10.1039/d4cc01656d -
The Plant Cell May 2024Transcription of antisense long noncoding RNAs (lncRNAs) occurs pervasively across eukaryotic genomes. Only a few antisense lncRNAs have been characterized and shown to...
Transcription of antisense long noncoding RNAs (lncRNAs) occurs pervasively across eukaryotic genomes. Only a few antisense lncRNAs have been characterized and shown to control biological processes, albeit with idiosyncratic regulatory mechanisms. Thus, we largely lack knowledge about the general role of antisense transcription in eukaryotic organisms. Here, we characterized genes with antisense transcription initiating close to the Poly(A) signal (PAS genes) in Arabidopsis (Arabidopsis thaliana). We compared plant native elongation transcript sequencing (plaNET-seq) with RNA sequencing (RNA-seq) during short-term cold exposure and detected massive differences between the response in active transcription and steady-state levels of PAS gene-derived mRNAs. The cold-induced expression of transcription factors B-BOX DOMAIN PROTEIN28 (BBX28) and C2H2-TYPE ZINC FINGER FAMILY PROTEIN5 (ZAT5) was detected by plaNET-seq, while their steady-state level was only slightly altered due to high mRNA turnover. Knockdown of BBX28 and ZAT5 or of their respective antisense transcripts severely compromised plant freezing tolerance. Decreased antisense transcript expression levels resulted in a reduced cold response of BBX28 and ZAT5, revealing a positive regulatory role of both antisense transcripts. This study expands the known repertoire of noncoding transcripts. It highlights that native transcription approaches can complement steady state RNA techniques to identify biologically relevant players in stress responses.
PubMed: 38801743
DOI: 10.1093/plcell/koae160 -
RNA Biology Jan 2024Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant...
Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant challenge to characterize them from a biological sample. In this study, we assessed the outcomes of bovine circRNA identification using six enrichment approaches with the combination of ribosomal RNAs removal (); linear RNAs degradation (); linear RNAs and RNAs with structured 3' ends degradation (); ribosomal RNAs coupled with linear RNAs elimination (); ribosomal RNA, linear RNAs and RNAs with poly (A) tailing elimination (); and ribosomal RNA, linear RNAs and RNAs with structured 3' ends elimination (), respectively. RNA-sequencing analysis revealed that different approaches led to varied ratio of uniquely mapped reads, false-positive rate of identifying circRNAs, and the number of circRNAs per million clean reads ( <0.05). Out of 2,285 and 2,939 highly confident circRNAs identified in liver and rumen tissues, respectively, 308 and 260 were commonly identified from five methods, with Ribo-RTP method identified the highest number of circRNAs. Besides, 507 of 4,051 identified bovine highly confident circRNAs had shared splicing sites with human circRNAs. The findings from this work provide optimized methods to identify bovine circRNAs from cattle tissues for downstream research of their biological roles in cattle.
Topics: Cattle; RNA, Circular; Animals; RNA, Ribosomal; Sequence Analysis, RNA; Liver; Rumen; Computational Biology; Gene Expression Profiling; Humans
PubMed: 38797889
DOI: 10.1080/15476286.2024.2356334 -
Journal of Experimental Botany May 2024Limonium bicolor, known horticulturally as sea lavender, is a typical recretohalophyte with salt glands in its leaf epidermis that secrete excess Na+ out of the plant....
Global analysis of key post-transcriptional regulation in early leaf development of Limonium bicolor identifies a long non-coding RNA that promotes salt gland development and salt resistance.
Limonium bicolor, known horticulturally as sea lavender, is a typical recretohalophyte with salt glands in its leaf epidermis that secrete excess Na+ out of the plant. Although many genes have been proposed to contribute to salt gland initiation and development, a detailed analysis of alternative splicing, alternative polyadenylation patterns, and long non-coding RNAs (lncRNAs) has been lacking. Here, we applied single-molecule long-read mRNA isoform sequencing (Iso-seq) to explore the complexity of the L. bicolor transcriptome in leaves during salt gland initiation (stage A) and salt gland differentiation (stage B) based on the reference genome. We identified alternative splicing events and the use of alternative poly(A) sites unique to stage A or stage B, leading to the hypothesis that they might contribute to the differentiation of salt glands. Based on the Iso-seq data and RNA in situ hybridization of candidate genes, we selected the lncRNA Btranscript_153392 for gene editing and virus-induced gene silencing to dissect its function. In the absence of this transcript, we observed fewer salt glands on the leaf epidermis, leading to diminished salt secretion and salt tolerance. Our data provide abundant transcriptome resources for unraveling the mechanisms behind salt gland development and furthering crop transformation efforts towards enhanced survivability in saline soils.
PubMed: 38795330
DOI: 10.1093/jxb/erae241 -
NPJ Parkinson's Disease May 2024A biallelic (AAGGG) expansion in the poly(A) tail of an AluSx3 transposable element within the gene RFC1 is a frequent cause of cerebellar ataxia, neuropathy, vestibular...
A biallelic (AAGGG) expansion in the poly(A) tail of an AluSx3 transposable element within the gene RFC1 is a frequent cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS), and more recently, has been reported as a rare cause of Parkinson's disease (PD) in the Finnish population. Here, we investigate the prevalence of RFC1 (AAGGG) expansions in PD patients of non-Finnish European ancestry in 1609 individuals from the Parkinson's Progression Markers Initiative study. We identified four PD patients carrying the biallelic RFC1 (AAGGG) expansion and did not identify any carriers in controls.
PubMed: 38789445
DOI: 10.1038/s41531-024-00723-0 -
European Journal of Endocrinology Jun 2024A 29-year-old female, born to consanguineous parents, was found with unmeasurable levels of vitamin D (<10 nmol/L) after routine biochemical screening during her first...
A 29-year-old female, born to consanguineous parents, was found with unmeasurable levels of vitamin D (<10 nmol/L) after routine biochemical screening during her first pregnancy. She did not respond to either oral or intramuscular vitamin D supplementation and was an otherwise healthy young woman, with no signs of rickets, osteomalacia, osteoporosis, or secondary hyperparathyroidism. Western blot analysis revealed total lack of vitamin D binding protein, and next generation sequencing confirmed a novel, pathogenic homozygote loss-of-function mutation in exon 13 of the group-specific component gene, that encodes the poly A tail for vitamin D binding protein. She was therefore diagnosed with hereditary DBP deficiency, and vitamin D supplementation was diminished to life-long regular vitamin D supplementation (25 μg per day). This case is extremely interesting, as it expands our knowledge of vitamin D physiology and supports the free hormone hypothesis, given that the patient was asymptomatic despite no measurable levels of vitamin D.
Topics: Humans; Female; Adult; Vitamin D; Vitamin D Deficiency; Vitamin D-Binding Protein; Homozygote; Loss of Function Mutation
PubMed: 38788201
DOI: 10.1093/ejendo/lvae061